Megan R. Edwards

Ebola Virus VP24 Targets a Unique NLS Binding Site on Karyopherin Alpha 5 to Selectively Compete with Nuclear Import of Phosphorylated STAT1.

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex with eVP24. In the complex, eVP24 recognizes a unique nonclassical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that may be required for viral replication unaffected.

Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen–associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.

The Marburg Virus VP24 Protein Interacts with Keap1 to Activate the Cytoprotective Antioxidant Response Pathway.

Kelch-like ECH-associated protein 1 (Keap1) is a ubiquitin E3 ligase specificity factor that targets transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for ubiquitination and degradation. Disrupting Keap1-Nrf2 interaction stabilizes Nrf2, resulting in Nrf2 nuclear accumulation, binding to antioxidant response elements (AREs), and transcription of cytoprotective genes. Marburg virus (MARV) is a zoonotic pathogen that likely uses bats as reservoir hosts. We demonstrate that MARV protein VP24 (mVP24) binds the Kelch domain of either human or bat Keap1. This binding is of high affinity and 1:1 stoichiometry and activates Nrf2. Modeling based on the Zaire ebolavirus (EBOV) VP24 (eVP24) structure identified in mVP24 an acidic loop (K-loop) critical for Keap1 interaction. Transfer of the K-loop to eVP24, which otherwise does not bind Keap1, confers Keap1 binding and Nrf2 activation, and infection by MARV, but not EBOV, activates ARE gene expression. Therefore, MARV targets Keap1 to activate Nrf2-induced cytoprotective responses during infection.

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation

Unwinding DNA and unleasing inflammation Fighting infections often comes with collateral damage, which sometimes can be deadly. For instance, in septic shock, the overwhelming release of inflammatory mediators drives multi-organ failure. Rialdi et al. now report a potential new therapeutic target for controlling excessive inflammation: the DNA unwinding enzyme topoisomerase I (Top1) (see the Perspective by Pope and Medzhitov). Upon infection, Top1 specifically localizes to the promoters of pathogen-induced genes and promotes their transcription by helping to recruit RNA polymerase II. Pharmacological inhibition of Top1 in a therapeutic setting increased survival in several mouse models of severe microbially induced inflammation. Science, this issue p. 10.1126/science.aad7993; see also p. 1058 Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses, as well as bacterial products. INTRODUCTION Infection causes inflammation, which contributes to pathogen clearance and organismal survival. The balance between the intensity and resolution of an inflammatory response is key for the fitness of the organism. Sepsis, for example, is a life-threatening condition caused by an excessive host response to infection, which in turn leads to multi-organ failure and death. Worldwide, millions of people each year succumb to sepsis. With an overall mortality rate of 20 to 50%, sepsis is the 10th leading cause of death (more than HIV and breast cancer) in the United States, according to the Centers for Disease Control and Prevention. Estimates indicate that 250,000 to 500,000 people die from sepsis annually in the United States. Children and the elderly are especially vulnerable to sepsis; it is the most common cause of death in infants and children. Childhood pneumonia, often caused by virus-bacteria co-infection, leads to septic shock and lung destruction. This occurs after bacterial invasion even in the presence of an appropriate antibiotic therapy. Finding remedies to treat sepsis and diseases associated with detrimental acute inflammatory reactions is thus pivotal for humankind. RATIONALE We reasoned that if excessive inflammation in response to infection leads to lethal consequences, dampening inflammation could be advantageous for the host. At least two strategies could be used to suppress inflammatory responses associated with infection. One is indirect and targets the pathogen (antibiotics). The second one, which we used, directly acts on the host response itself. In such a strategy, the suppression of acute inflammation would bypass the fatal outcome associated with overt inflammation and would “buy time” to allow the host immune response to eliminate the pathogen. After microbial invasion, many steps could be targeted between the early phases of the cellular response (sensing of the pathogen and signal transduction) and the information flow from DNA to RNA to proteins that act as inflammatory mediators (i.e., cytokines). We decided to identify and chemically inhibit cellular factors that act at the DNA (chromatin) level and play a primary role in activating the expression of inflammatory genes. RESULTS We found that chemical inhibition of topoisomerase 1 (Top1), an enzyme that unwinds DNA, suppresses the expression of infection-induced genes with little to no effect on housekeeping gene expression and without cellular damage. In vitro, depletion or chemical inhibition of Top1 in epithelial cells and macrophages suppresses the host response against influenza and Ebola viruses as well as bacterial products. At the mechanistic level, as shown by chemical genetics and epigenetic approaches, Top1 inhibition primarily suppresses RNA polymerase II (RNAPII) activity at pathogen-associated molecular pattern (PAMP)–induced genes. These genes require SWI/SNF chromatin remodeling for activation and display unique genetic and epigenetic features, such as the presence of IRF3 binding sites, low basal levels of RNAPII, histone H3 Lys27 acetylation marks, DNA hypersensitivity, and CpG islands. This gene “signature” of specificity was also validated using public data sets. In vivo, Top1 inhibition therapy rescued 70 to 90% mortality caused by exacerbated inflammation in three mouse models: acute bacteria infection, liver failure, and virus-bacteria co-infection. Strikingly, one to three doses of inhibitors were sufficient for the protective effect in all models, without overt side effects. CONCLUSION The inflammatory immune response against microbes is essential in protecting us against infections. In some cases, such as highly pathogenic and pandemic infections, the organism turns against itself and responds too acutely, with an excessive inflammation that can have fatal consequences. Our results suggest that a therapy based on Top1 inhibition could save millions of people affected by sepsis, pandemics, and many congenital deficiencies associated with acute inflammatory episodes and “cytokine storms.” CREDIT: RYGER/SHUTTERSTOCK The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response.

Deep Sequencing Identifies Noncanonical Editing of Ebola and Marburg Virus RNAs in Infected Cells

ABSTRACT Deep sequencing of RNAs produced by Zaire ebolavirus (EBOV) or the Angola strain of Marburgvirus (MARV-Ang) identified novel viral and cellular mechanisms that diversify the coding and noncoding sequences of viral mRNAs and genomic RNAs. We identified previously undescribed sites within the EBOV and MARV-Ang mRNAs where apparent cotranscriptional editing has resulted in the addition of non-template-encoded residues within the EBOV glycoprotein (GP) mRNA, the MARV-Ang nucleoprotein (NP) mRNA, and the MARV-Ang polymerase (L) mRNA, such that novel viral translation products could be produced. Further, we found that the well-characterized EBOV GP mRNA editing site is modified at a high frequency during viral genome RNA replication. Additionally, editing hot spots representing sites of apparent adenosine deaminase activity were found in the MARV-Ang NP 3′-untranslated region. These studies identify novel filovirus-host interactions and reveal production of a greater diversity of filoviral gene products than was previously appreciated. IMPORTANCE This study identifies novel mechanisms that alter the protein coding capacities of Ebola and Marburg virus mRNAs. Therefore, filovirus gene expression is more complex and diverse than previously recognized. These observations suggest new directions in understanding the regulation of filovirus gene expression. This study identifies novel mechanisms that alter the protein coding capacities of Ebola and Marburg virus mRNAs. Therefore, filovirus gene expression is more complex and diverse than previously recognized. These observations suggest new directions in understanding the regulation of filovirus gene expression.

High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication

Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.

Marburg Virus VP24 Protein Relieves Suppression of the NF–κB Pathway Through Interaction With Kelch-like ECH-Associated Protein 1

BACKGROUND Marburg virus (MARV) is an emerging zoonotic pathogen that causes hemorrhagic fever. MARV VP24 (mVP24) protein interacts with the host cell protein Kelch-like-ECH-associated protein 1 (Keap1). Keap1 interacts with and promotes the degradation of IκB kinase β (IKKβ), a component of the IκB kinase (IKK) complex that regulates nuclear factor-κB (NF-κB) activity. We studied whether mVP24 could relieve Keap1 repression of the NF-κB pathway. METHODS Coimmunoprecipitation assays were used to examine the interaction between Keap1 and IKKβ in the presence of wild-type mVP24 and mutants of mVP24 defective for binding to Keap1. Western blotting was used to determine levels of IKKβ expression in the presence of Keap1 and mVP24. NF-κB promoter-luciferase assays were used to determine the effect of mVP24 on Keap1-induced repression of activity. RESULTS Expression of wild-type mVP24 disrupted the interaction of IKKβ and Keap1, whereas weakly interacting and noninteracting mVP24 mutants did not disrupt the interaction between Keap1 and IKKβ. The interaction of mVP24 with Keap1 enhanced IKKβ levels in the presence of Keap1. The interaction of mVP24 with Keap1 also relieved Keap1 repression of NF-κB reporter activity. CONCLUSIONS mVP24 can relieve Keap1 repression of the NF-κB pathway through its interaction with Keap1.

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

ABSTRACT Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCE The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication

Abstract Ebola virus (EBOV) is an enveloped negative‐sense, single‐stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus‐encoded complex with RNA‐dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure‐activity relationship (SAR) studies of the benzoquinoline scaffold produced ˜50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC50 in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative‐sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive‐sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti‐EBOV compounds and identifies the benzoquinoline series as a broad‐spectrum antiviral lead. HighlightsAn Ebola virus minigenome assay and a novel counterscreen were used to screen a 200,000 compound library.The screen identified a benzoquinoline scaffold as an inhibitor of Ebola virus RNA synthesis.SAR studies indicate that the full three‐ring structure of the heterocycle is essential for inhibition of the minigenome assay.An optimized compound, SW456, exhibits broad spectrum activity against filoviruses, vesicular stomatitis virus and Zika virus.